mRNA in Lipid Nanoparticles (LNPs) and Liposomes

LNP illustration

In-vitro mRNA manufacturing technology has enabled rapid vaccine development and supply in response to the COVID-19 pandemic. Furthermore, therapeutic applications have been under investigation since many years. For drug administration, mRNA technology requires lipid nanoparticles (LNPs) or other types of liposomes to encapsulate and protect the mRNA during delivery of the genetic information into patient cells.

mRNA quality can be assessed by standard analytical techniques (electrophoresis or chromatography), while identity needs to be assured by sequencing. Following lipid nanoparticle formulation and drug product manufacturing, the characterization of the resulting polydisperse nanoparticle mixture and its homogeneous loading with mRNA require extended characterization techniques in order to demonstrate and validate the robustness of the manufacturing process and drug product.

Analytical ultracentrifugation is the standard technology for the analysis of these mixtures. Their complexity is countervailed by the combination of orthogonal AUC techniques, such as sedimentation velocity and density variation experiments. Nanolytics offers dedicated experience in differentiating empty (non-chromophoric) and cargo-loaded (chromophoric) liposomes by exploiting our unique multiplex detection capabilities (4D AUC and AIDA). Distinct LNP/liposome compositions, in particular the cargo type such as complete and fragmented mRNA, are verified via combination of hydrodynamic and spectroscopic properties from a single sedimentation velocity experiment. Conclusions can be optionally corroborated by density gradient ultracentrifugation providing buoyant densities combined with the spectroscopic profile.

CASE STUDY: Peptide-loaded liposomes with a low payload ratio

As a major challenge, both the direct detection of empty liposomes and loaded liposomes is constrained to interference measurement.
This does not allow to spectroscopically obtain the payload content. Furthermore, density gradients may not be applicable as high salt content may affect the liposomes; also, their density is too low for typical gradients. The course of action taken in the AAV case study is not feasible.

Alternatively, density variation of the solvent can be applied. Exploiting differences in particle density due to payload ratio is an important approach, as particle density increases with payload content. Two sedimentation velocity experiments, diluting the sample with deuterium oxide, can be correlated, yielding a particle density distribution. In this example, the effect is easy to see via the presence of floating (low payload content) and sedimenting material (high payload content) at high solvent density, wheras all material sediments at low solvent density.